Results SAA levels were increased in the inflamed group compared with the HFD and control groups, suggesting that inflammation was successfully induced. There were no differences in body weight among three groups at each week. Interestingly, inflammation significantly reduced serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels compared with the HFD mice. However, both foam cell formation in cardiac blood vessels and cardiac collagen deposition were increased in the inflamed group, as demonstrated by HE and Masson trichrome staining. Furthermore, inflammation reduced protein expression of CD31 and increased protein expression of alpha-smooth muscle actin (α-SMA) and collagen I, which contribute to cardiac EndMT.

Introduction

Cardiac fibrosis is a common feature in patients with advanced cardiac failure regardless of the aetiology of cardiomyopathy [1]. Cardiac fibrosis, which is associated with decreased microvasculature and the disruption of normal myocardial structure, results from an excessive deposition of extracellular matrix, which is mediated by the recruitment of fibroblasts. However, the origin of these fibroblasts in the adult heart is unclear [2]. Traditionally, adult fibroblasts are derived directly from embryonic mesenchymal cells and increase in number only because of the proliferation of resident fibroblasts. Recent studies have suggested that during fibrosis, bone marrow-derived fibroblasts and endothelial cells contribute to fibroblast accumulation through endothelial-mesenchymal transition (EndMT) in addition to the proliferation of resident fibroblasts [3, 4]. During EndMT, resident endothelial cells delaminate from an organised cell layer and invade the underlying tissue. This mesenchymal phenotype is characterised by reduced expression of endothelial markers, such as CD31 or vascular endothelial cadherin, and increased expression of mesenchymal markers, such as fibroblast-specific protein-1, collagen I, or α-smooth muscle actin (α-SMA), as well as the loss of cell-cell junctions and the acquisition of invasive and migratory properties. Thus, EndMT-derived cells function as fibroblasts in damaged tissue and may have an important role in tissue remodelling and fibrosis [5-8].

Lipid profile assessment

Tissue processing

Heart sections were rinsed with saline and placed in 10% buffered formalin. After treatment, representative sections were embedded in paraffin.

Haematoxylin-eosin (HE) Staining

Paraffin-embedded hearts were sectioned and dewaxed. Sections were stained for 15 minutes with haematoxylin, followed by 1% eosin for 3 minutes. After dehydration, resinene was used to seal the sections to transparency. The sections were observed under light microscopy (×400).

Statistical analysis

All of the data are expressed as the mean ± standard deviation (SD) and were processed with SPSS software 13.0. A one-Way ANOVA was used to compare continuous variables among the three groups where appropriate. Differences were considered to be significant if the P value was less than 0.05.

Results

We induced inflammatory atherosclerosis in ApoE KO mice by injecting them with 10% casein. We found that serum SAA was significantly increased in the casein-injected mice compared with the controls, suggesting that systemic inflammation was successfully induced (Figure 1).

There were no differences in body weight change among three groups at each week

To exclude the effect of body weight on serum levels of total cholesterol, triglyceride LDL, and HDL, we measured the body weight of ApoE KO mice, and found that there were no differences in body weight change at each week among three groups (Figure 2).

As shown in Figure 3, there were significantly increased serum total cholesterol, triglyceride and LDL levels in high-fat-fed ApoE KO mice compared with the controls. However, serum total cholesterol, triglyceride and LDL levels were decreased in the casein-injected ApoE KO mice compared with the HFD mice (Figure 3).

Using HE and Masson's trichrome staining, we determined that the high-fat diet induced foam cell formation in cardiac blood vessels (Figure 4A) and cardiac collagen deposition compared with the control (Figure 4B, 4C). Meanwhile, immunohistochemistry demonstrated that collagen I protein expression was significantly increased in the HFD and inflamed groups compared with the control (Figure 4D). Interestingly, although the serum total cholesterol, triglyceride, and LDL levels in the inflamed group were lower than in the HFD group, the degree of foam cell formation and cardiac fibrosis in the inflamed group was enhanced compared with the HFD group.

Body weight change at each week (n=8). ApoE KO mice were fed normal chow (Control) or Western diet for eight weeks without (HFD group) or with 10% casein injections (Inflamed group). The body weight change was measured at each week.

To assess the possible mechanisms of hyperlipidaemia and inflammation-induced cardiac fibrosis, we evaluated the effects of inflammation on the protein expression of EndMT biomarkers collagen I, CD31 and α-SMA in the ApoE KO mouse hearts. Western blot demonstrated that inflammation significantly increased the expression of collagen I and α-SMA but decreased CD31 protein expression in high-fat-fed ApoE KO mice (Fig 5A and 5B). These data suggest that inflammation exacerbates the progression of dyslipidemia-induced cardiac fibrosis via EndMT.

Discussion

Cardiac fibrosis is a multi-factorial disease that occurs in several pathological processes, including hypertension, diabetes, radiation, viral myocarditis, and genetic mutations [16]. Inflammatory stress and dyslipidaemia synergise to cause organ injuries [13-15, 17]. Thus, this study investigated the role of both inflammatory stress and dyslipidaemia in cardiac fibrosis, as well as possible mechanisms for their synergy, using ApoE KO mice.

We subcutaneously injected the high-fat-fed ApoE KO mice with 10% casein and successfully induced inflammatory atherosclerosis, as shown by significantly increased plasma SAA levels compared with the HFD group. These results demonstrated that inflammatory stress markedly exacerbated lipid accumulation in cardiac blood vessels and cardiac collagen deposition, which contributed to the progression of cardiac fibrosis. Interestingly, serum total cholesterol, triglyceride and LDL levels in the casein-injected mice were lower than in the HFD group. In order to exclude the factors from reduced food intake or increased fecal excretion, we checked the body weight in three groups of mice. As shown by Figure 2, there were no differences about body weight change at each week among three groups. The results suggest that the balance of food intake and fecal excretion in casein injection group was roughly equivalent to that in the control and HFD group. Our previous in vivo and in vitro studies demonstrated that inflammatory stress disrupted cholesterol homeostasis through dysregulation of the LDLr pathway and exacerbated the progression of atherosclerosis, vascular calcification, and liver injury [14, 18, 19]. Therefore, inflammatory stress may induce lipid redistribution from the circulation to the cardiac tissues, thereby causing hypolipidaemia and cardiac fibrosis [20].

To explore potential mechanisms, we further assessed the effects of dyslipidaemia and inflammatory stress on EndMT in ApoE KO mouse hearts. Our results demonstrated that hyperlipidaemia upregulated the protein expression of collagen I and α-SMA but decreased the expression of CD31, interestingly, which were deteriorated by inflammation. These data suggest that inflammatory stress and hyperlipidaemia in combination contribute to the progression of cardiac fibrosis via EndMT.

Dyslipidaemia was also strongly associated with the progression of cardiac fibrosis [9]. Qin et al. demonstrated that simvastatin inhibited cardiac hypertrophy and fibrosis by reducing total cholesterol and matrix metalloproteinase-9 levels in Western diet-fed Apolipoprotein E-deficient mice [27]. Wendt et al. suggested a link between hypercholesterolemia and left ventricular fibrosis in Dahl salt-sensitive rats, which are genetically modulated by chromosome 19 [28].

In summary, our findings demonstrated for the first time that inflammatory stress exacerbates the progression of cardiac fibrosis in high-fat-fed ApoE KO mice via EndMT, suggesting that hyperlipidaemia and inflammation synergise to redistribute lipid from the plasma to the heart, thereby accelerating the progression of cardiac fibrosis.

Acknowledgements

This work was supported by grants 81070571 and 81170792 from the National Natural Science Foundation of China.